Reversible inhibition in bimolecular rapid-equilibrium random-order enzyme systems. The effect of substrate–substrate and inhibitor–substrate interactions

Clark, Andy

doi:10.1042/bj1170997

Cited by 7 publications

(3 citation statements)

References 9 publications

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“…Mg2+ was maintained in excess at 6.75 mm in order to ensure full binding of Mg2+ to ATP, which did not exceed 1 mm. All determinations were performed under conditions of limiting concentrations of both substrates (glucose and MgATP2-) (Cleland, 1963b;Clark, 1970). Kinetic results were analysed by regression lines where relevant, calculated by the method of least squares.…”

Section: Methodsmentioning

confidence: 99%

“…both types of mechanism will conformn to the equation below in the absence of inhibitors (Clark, 1970).…”

Section: Ag/kg -435 Rkag/kya -Agkmentioning

confidence: 99%

“…In contrast, with a random rapid equilibrium mechanism, fully competitive inhibition with respect to either substrate will produce similar plotting patterns. Double-reciprocal plots made with respect to the competing substrate will be competitive in char-acter, whereas those made with respect to the non-competing substrate will possess a common intersection point above, below or on the abscis,sa, if K, (slope) is smaller than, greater than or equal to K, (intercept), depending on the degree of subsitrate-substrate and inhibitor-substrate interaction (Cleland, 1963b;Clark, 1970). The initialvelocity equations will then be:…”

Section: Ag/kg -435 Rkag/kya -Agkmentioning

confidence: 99%

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Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

Bachelard

Clark

Thompson

1971

Biochemical Journal

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1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.

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Section: Methodsmentioning

confidence: 99%

“…both types of mechanism will conformn to the equation below in the absence of inhibitors (Clark, 1970).…”

Section: Ag/kg -435 Rkag/kya -Agkmentioning

confidence: 99%

Section: Ag/kg -435 Rkag/kya -Agkmentioning

confidence: 99%

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